Split-process recombine methods in combinatorial chemistry are already known in relation to formation of peptide libraries as discussed in Gallop et al., 1994, J. Med. Chem. 37 1233-1251 which refers to synthesis of peptide libraries by the use of polystyrene beads which are initially present as a first batch which are split into smaller batches wherein different amino acids are covalently attached to a primary linker group present on the surface of each bead. Subsequently, the beads are recombined and then split again so that a second amino acid may be attached to the amino acid attached to the primary linker group. This process is repeated a number of times as may be required to produce the peptide library.
A similar procedure is described in Gallop et al., 1994, supra which refers to the establishment of an oligonucleotide library.
“Split-process-recombine” or “split synthesis” methods generating one (resin) bead-one compound libraries were first proposed in Furka et al., 1991, Int. J. Pept. Protein Res. 37 487-493 and are also discussed in Eichler et al., 1995, Medicinal Research Reviews 15(6) 481-496 and Balkenhohl et al., 1996, Angew. Chem. Int. Ed. Engl. 35 2288-2337.
Peptide libraries are mainly used in drug discovery as discussed in Gallop et al., 1994, supra wherein potentially useful drugs are identified by screening methods as are known in the art. This is also reported in Borman Chemical & Engineering News, February 1997, 43-62, Fruchtel et al., 1996, Angew. Chem. Int. Ed. Engl. 35 17-42 and Barany et al., 1987, In. J. Peptide Protein Res. 30 705-739.
Oligonucleotide libraries, on the other hand, are useful as a tool for rapid DNA sequencing by hybridization as discussed in Fodor et al., 1991, Science 251 767, Lysov et al., 1988, Dokl. Akad. Nauk. SSSR 303 1508, Bains et al., 1988, J. Theor. Biol. 135 303, Drmanac et al., 1989, Genomics 4 114 and Drmanac et al., 1993, Science 260 1649.
Sequencing by hybridization (SBH) has been proposed to replace conventional DNA sequencing technology which is a laborious procedure involving electrophoretic size separation of labelled DNA fragments. SBH uses a set of short oligonucleotide probes of defined sequence to search for complementary sequences on a longer target strand of DNA. The hybridization pattern is used to reconstruct the target DNA sequence.
The challenge with implementing SBH techniques as a viable method of sequencing of DNA is that an extremely large number of probes is required. New methods have been proposed to overcome this problem as discussed in Fodor et. al., 1991, supra, Pease et al., 1994, Proc. Natl. Acad. Sci. 91 5022, Cho et al., 1993, Science 261 1303 and Southern et al., 1992, Genomics 13 1008. These new methods involve the use of oligonucleotide arrays or “biological chips” as discussed in Fodor et al., 1991, supra, which harbour specified chemical compounds (i.e. the probes) at precise locations in an array format. The target DNA is then added to the array of probes. The hybridization pattern, determined in a single experiment, directly reveals the identity of all complementary probes as reported in Drmanac et al., 1989, supra and Drmanac et al., 1993, supra. Although this technique holds much promise, the information density on each array is extremely low for the purpose of DNA sequencing and this limits the size and speed with which DNA fragments can be sequenced. The difficulties associated with selectively anchoring oligonucleotide sequences to specific and spatially arranged sites on the substrate means that the minimum pixel size in the arrays is limited currently to approximately 0.4 mm×0.4 mm in area. As pixel size directly determines information density and hence sequencing efficiency, miniaturization of the “biological chips” is a major technical problem for implementing this technology as a rapid method of sequencing. One method of overcoming this problem is the use of a technique requiring “field induced colloidal crystallization” as reported in Trau et al., 1996, Science 272 706. This technique uses miniaturized chips of patterned microscopic colloidal particles which contain chemisorbed oligonucleotides on a transparent electrode comprising indium tin oxide. Fluorescent hybridization patterns of unknown DNA sequences with the arrays are observed using an optical microscope.
Before the advent of the technique of Trau et al., 1996, supra, SBH was previously carried out by attaching target DNA to a surface and sequentially interrogating with a set of oligonucleotide probes, one at a time as discussed in Drmanac et al., 1989, supra, Drmanac et al., 1993, supra and Strezoska et al., 1991, Proc. Nat. Acad. Sci. USA 88 10089 which was time consuming and inefficient.
Application of conventional split-process-recombine methods to drug discovery and SBH is, however, currently limited by the inherent difficulty of rapidly, and conveniently, identifying the unique sequence of events applicable to any chosen multimeric molecule. For large numbers of carriers and large numbers of steps and/or processing methods, this “identification” procedure is particularly difficult. In many practical cases, where high throughput and fast analysis is required, this problem is intractable by conventional methods.
The conventional split-process-recombine technologies referred to above presented difficulties when it was desired to detect and isolate a molecule of interest. In this regard, it was necessary to detect the molecule of interest by use of a suitable assay or probe and then isolate the molecule of interest by cleaving that molecule from the bead and subsequently identifying the molecule by techniques such as mass spectroscopy or HPLC. This was time consuming and cumbersome and in some cases, cleavage was not possible.
Reference may be made to International Publication WO93/06121 which refers to a general stochastic method for synthesizing libraries of random oligomers, which are synthesized on solid supports inclusive of polystyrene beads or which may be cleaved from these supports to provide a soluble library. The oligomers are composed of a sequence of monomers that can be joined together to form an oligomer or polymer. This reference also describes the use of identifier tags to identify the sequence of monomers in the oligomer. The identifier tag may be attached directly to the oligomer with or without an accompanying particle, to a linker attached to the oligomer, to the solid phase support on which the oligomer is synthesized or to a second particle attached to the oligomer carrying particle. However, the only means of attachment described in this reference is by way of covalent bonding. In this reference, the identifier tag is described in very broad terms, such as any recognizable feature, which includes a microscopically distinguishable shape, size, colour or optical density; a differential absorbance or emission of light; chemical reactivity; magnetic or electronic coiled information; or any other distinctive mark with the required information and decipherable at the level of one or a few solid supports. In one form, the identifier tags are described as small beads of recognizably different shapes, sizes or colours or labelled with bar codes.
However, while the description of International Publication WO93/06121 refers very broadly to the types of identifier tags that may be utilized in the method of formation of oligomer libraries, the only experimental evidence referred to in the specification is the use of oligonucleotides. Thus, there is no enabling disclosure especially in relation to the use of small beads as identifier tags and how this particular technique may be put into practical effect.
In International Publication WO93/06121, reference is made to identifying the tags by sequencing or hybridization if the tag is an oligonucleotide. One can also amplify the oligonucleotide tag by PCR. However, it will be appreciated that such identification methods are time consuming and inefficient. For example, use of PCR may result in PCR product contamination making it necessary to introduce further measures to overcome this problem as described in International Publication No. WO93/06121. It is also necessary to sequence amplified DNA and this involves an additional step in the identification procedure as described in International Publication No. WO93/06121.
Reference may also be made to U.S. Pat. No. 5,721,099 which describes complex combinatorial chemical libraries of compounds encoded with tags. Each compound in the library is produced by a single reaction series and is bound to an individual solid support which may include particles or beads inclusive of polystyrene beads or silica gel beads. Each solid support has bound to it a combination of four distinguishable identifiers which differ from one another. The combination provides a specific formula comprising a tag component capable of analysis and a linking component capable of being selectively cleaved to release the tag component. Each identifier or combination thereof encodes information at a particular stage in the reaction series for the compound bound to the solid support. However, it is essential in this library that prior to analysis, each tag component must be cleaved from the support thus creating at least one additional step which is time consuming and inefficient and thus the same disadvantages relevant to International Publication WO93/06121 also apply in the case of this reference.
In relation to using single stranded identifier tags to encode combinatorial peptide synthesis, which method is discussed in Needles et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90 10700-10704, such method was disadvantageous because of the reasons discussed above in International Publication WO93/06121. However, it is also noted that after detection of a peptide or molecule within the library of interest, in some cases it was necessary to cleave the corresponding tag from the support and amplify the tag by PCR because it was only present in trace amounts. This was also time consuming and inefficient.
Reference may also be made to photolabile or electrophoretic tagging as described in Ohlmeyer et al., 1993, Proc. Nat. Acad. Sci. USA 90 10922-10926 or Gallop et al., 1994, supra which was also disadvantageous because of the inclusion of additional steps prior to identification of the tag.